No donors were recently shown to increase basal levels of both cGMP and cAMP in rat cardiac myocytes. We have shown that NO activates both PKG and PKA pathways with opposing contractile effects. Here we examined whether NO modulates cAMP/PKA signaling primarily via guanylate cyclase (GC) activation/cGMP generation (e.g., via cGMP-stimulated (cGS-) PDE) or by cGMP/PKG-independent mechanisms. In electrically stimulated, indo-1 loaded adult rat cardiac myocytes the NO donor S-nitroso N-acetyl-penicillamine (SNAP, 0.1 mM) significantly attenuated contraction (TA;-15plus/minus 4%), with a small, but significant increase in the Ca2+ transient (CaT;7plus/minus 3%). In contrast, cells pretreated with the GC-inhibitor, LY83583 (10mu M), demonstrated large increases in TA (39plus/minus5%) and CaT (31plus/minus4%) after SNAP, effects reversed by the PKA/PKG inhibitor H-7. Addition of SNAP to cells pretreated with equipotent (approximately TA-doubling) doses of the PDE-inhibitor IBMX (0.1 mM) and of norepinephrine (NE, 50 nM), resulted in significant increases in TA and CaT above IBMX alone, but with little change from NE alone. We conclude that: 1) NO increases cAMP production which is largely offset when GC is competent to produce cGMP, because of enhanced cAMP consumption via cGS-PDE. Thus, NO produces only modest PKA activation except when cGMP production, or cGS-PDE activity, is inhibited. 2) NO activates adenylate cyclase (AC) and PKA by novel mechanisms in rat cardiac myocytes independently of GC/cGMP activation, because of the positive inotropic effect of NO during GC inhibition. We propose that NO may activate G-proteins (G(s)) in cardiac myocytes, and is responsible for the increased AC/cAMP/PKA activity.